How do small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) regulate gene expression and protect the genome? RNAi encompasses the cellular response to exogenous double-stranded RNA (dsRNA)-delivered experimentally or generated during a viral infection - and the response to parasitic genetic eleiTients, such as transposons and highly repeated sequences. Our experimental strategy uses Drosophila melanogaster as a model system, because flies combine outstanding genetic, biochemical, and phenotypic tools. Small silencing RNAs, including siRNAs, microRNAs, and piRNAs, associate with members of the Argonaute family of proteins to form the RNA-induced silencing complex (RISC), which mediates RNAi and related silencing phenomena. We will use new technologies to study the function of RISC, including single-molecule fluorescence techniques to define the .shared and divergent properties of Argonaute proteins from flies, mice, and bacteria. Our goal is to understand how the specific biochemical properties of Argonaute proteins reflect their distinct biological roles. The siRNA pathway defends flies against exogenous pathogens-viruses, whereas the piRNA pathway controls endogenous pathogens - transposons. To better understand the role of the siRNA pathway in viral defense, we have developed a novel strategy in which flies become infected with viruses by environmental exposure. These studies will help us understand the complex response to different viruses and different viral doses. The studies will combine genetics, high throughput sequencing, and bioinformatic analyses. Finally, we seek to understand how the piRNA pathway defends the genome against transposons and repetitive sequences. To facilitate these efforts, we have developed a novel piRNA silencing system in which GFP inserted into a source of piRNAs silences an EGFP transgene inserted elsewhere in the genome, These new genetic tools promise to help us learn how piRNAs are produced and how they suppress transposon expression.